Versatile Site-Specific Conjugation of Small Molecules to siRNA Using

Feb 7, 2011 - We have previously demonstrated that conjugation of small molecule ligands to small interfering RNAs (siRNAs) and anti-microRNAs results...
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Versatile Site-Specific Conjugation of Small Molecules to siRNA Using Click Chemistry Takeshi Yamada,†,‡,§ Chang Geng Peng,†,§ Shigeo Matsuda,† Haripriya Addepalli,† K. Narayanannair Jayaprakash,† Md. Rowshon Alam,† Kathy Mills,† Martin A. Maier,† Klaus Charisse,† Mitsuo Sekine,‡ Muthiah Manoharan,*,† and Kallanthottathil G. Rajeev*,† †

‡

Drug Discovery, Alnylam Pharmaceuticals, Cambridge, Massachusetts 02142, United States, and Department of Life Science, Tokyo Institute of Technology, J2-12, 4259 Nagatsuta, Midoriku, Yokohama 226-8501, Japan. §Takeshi Yamada and Chang Geng Peng contributed equally to this work. [email protected] [email protected] Received September 26, 2010

We have previously demonstrated that conjugation of small molecule ligands to small interfering RNAs (siRNAs) and anti-microRNAs results in functional siRNAs and antagomirs in vivo. Here we report on the development of an efficient chemical strategy to make oligoribonucleotide-ligand conjugates using the copper-catalyzed azide-alkyne cycloaddition (CuAAC) or click reaction. Three click reaction approaches were evaluated for their feasibility and suitability for high-throughput synthesis: the CuAAC reaction at the monomer level prior to oligonucleotide synthesis, the solutionphase postsynthetic “click conjugation”, and the “click conjugation” on an immobilized and completely protected alkyne-oligonucleotide scaffold. Nucleosides bearing 50 -alkyne moieties were used for conjugation to the 50 -end of the oligonucleotide. Previously described 20 - and 30 -O-propargylated nucleosides were prepared to introduce the alkyne moiety to the 30 and 50 termini and to the internal positions of the scaffold. Azido-functionalized ligands bearing lipophilic long chain alkyls, cholesterol, oligoamine, and carbohydrate were utilized to study the effect of physicochemical characteristics of the incoming azide on click conjugation to the alkyne-oligonucleotide scaffold in solution and on immobilized solid support. We found that microwave-assisted click conjugation of azido-functionalized ligands to a fully protected solid-support bound alkyne-oligonucleotide prior to deprotection was the most efficient “click conjugation” strategy for site-specific, high-throughput oligonucleotide conjugate synthesis tested. The siRNA conjugates synthesized using this approach effectively silenced expression of a luciferase gene in a stably transformed HeLa cell line.

Introduction The so-called click chemistry strategy allows efficient coupling of modular building blocks bearing azide and alkyne.1

Due to improvements independently made to the Huisgen 1,3-dipolar azide-alkyne cycloaddition reaction2 by the laboratories of Meldal3 and Sharpless,1,4 the click reaction

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can be performed in aqueous and nonaqueous solvent and on solid-support. 5 The inertness of azide and alkyne toward other functional groups such as amines, carboxylates, alcohols, thiols, and esters makes this strategy advantageous for selective postsynthetic chemical ligation of ligands to proteins, peptides, nucleic acids, carbohydrates, and polymers. Click chemistry has proven very useful to the oligonucleotide chemistry field. The copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction has been successfully used for ligation and cyclization of DNA using appropriately azido- and alkyne-functionalized DNA strands.6-8 The CuAAC reaction has also been extensively used for synthesis of modified nucleosides,9-13 carbohydrate,14-16 fluorophore,17-21 and lipophilic22 oligonucleotide conjugates; for synthesis of chimeras of peptide-DNA/PNA23 and oligonucleotides;12 and to explore G-quadruplex solution structures.24 A recent report from Morvan’s laboratory demonstrated a very efficient click chemistry approach for the synthesis of a dendrimeric oligonucleotide-glycoconjugate bearing 16 galactose moieties on its periphery.25 In addition, Best has reviewed the usefulness of azide-alkyne cycloaddition in bioorthogonal reactions for labeling of biomolecules, including proteins, viruses, sugars, nucleic acids, and lipids, and its impact on chemical biology.26 (1) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2001, 40, 2004–2021. (2) Huisgen, R. Angew. Chem. 1963, 75, 604–637. (3) Tornoe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67, 3057–3064. (4) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew. Chem., Int. Ed. 2002, 41, 2596–2599. (5) Amblard, F.; Cho, J. H.; Schinazi, R. F. Chem. Rev. 2009, 109, 4207– 4220. (6) El-Sagheer, A. H.; Brown, T. Chem. Soc. Rev. 2010, 39, 1388–1405. (7) Lietard, J.; Meyer, A.; Vasseur, J.-J.; Morvan, F. J. Org. Chem. 2008, 73, 191–200. (8) Pourceau, G.; Meyer, A.; Vasseur, J.-J.; Morvan, F. J. Org. Chem. 2009, 74, 6837–6842. (9) Chittepu, P.; Sirivolu, V. R.; Seela, F. Bioorg. Med. Chem. 2008, 16, 8427–8439. (10) Gramlich, P. M. E.; Wirges, C. T.; Gierlich, J.; Carell, T. Org. Lett. 2008, 10, 249–251. (11) Lucas, R.; Zerrouki, R.; Granet, R.; Krausz, P.; Champavier, Y. Tetrahedron 2008, 64, 5467–5471. (12) Jawalekar, A. M.; Meeuwenoord, N.; Cremers, J. G. O.; Overkleeft, H. S.; van der Marel, G. A.; Rutjes, F. P. J. T.; van Delft, F. L. J. Org. Chem. 2008, 73, 287–290. (13) Andersen, N. K.; Chandak, N.; Brulikova, L.; Kumar, P.; Jensen, M. D.; Jensen, F.; Sharma, P. K.; Nielsen, P. Bioorg. Med. Chem. 2010, 18, 4702–4710. (14) L€ onnberg, H. Curr. Org. Synth. 2009, 6, 400–425. (15) Zhang, J.; Pourceau, G.; Meyer, A.; Vidal, S.; Praly, J.-P.; Souteyrand, E.; Vasseur, J.-J.; Morvan, F.; Chevolot, Y. Biosens. Bioelectron. 2009, 24, 2515– 2521. (16) Kiviniemi, A.; Virta, P.; L€ onnberg, H. Bioconjugate Chem. 2010, 21, 1890–1901. (17) Seo, T. S.; Li, Z.; Ruparel, H.; Ju, J. J. Org. Chem. 2003, 68, 609–612. (18) Berndl, S.; Herzig, N.; Kele, P.; Lachmann, D.; Li, X.; Wolfbeis, O. S.; Wagenknecht, H.-A. Bioconjugate Chem. 2009, 20, 558–564. (19) Seela, F.; Ingale, S. A. J. Org. Chem. 2010, 75, 284–295. (20) Weisbrod, S. H.; Marx, A. Chem. Commun. 2008, 5675–5685. (21) Gramlich, P. M. E.; Warncke, S.; Gierlich, J.; Carell, T. Angew. Chem., Int. Ed. 2008, 47, 3442–3444. (22) Godeau, G.; Staedel, C.; Barthelemy, P. J. Med. Chem. 2008, 51, 4374–4376. (23) Gogoi, K.; Mane, M. V.; Kunte, S. S.; Kumar, V. A. Nucleic Acids Res. 2007, 35, e139/1; DOI: 10.1093/nar/gkm935. (24) Xu, Y.; Suzuki, Y.; Komiyama, M. Angew. Chem., Int. Ed. 2009, 48, 3281–3284. (25) Pourceau, G.; Meyer, A.; Chevolot, Y.; Souteyrand, E.; Vasseur, J.-J.; Morvan, F. Bioconjugate Chem. 2010, 21, 1520–1529. (26) Best, M. D. Biochemistry 2009, 48, 6571–6584.

JOC Featured Article In order to make small interfering RNAs (siRNAs) therapeutically active, the molecules must be delivered into the cytoplasm of the target cells, where the RNA-induced silencing complex (RISC) is located. Various approaches, such as chemical modifications, conjugation of small molecules, liposomes, nanoparticles, polymers, polyamines, and cellpenetrating peptides have been investigated to enhance cellular uptake.27-34 Conjugation of small molecules such as cholesterol, fatty acids, vitamin E, polycationic compounds, and receptor specific ligand like folic acid and carbohydrates can also improve pharmacokinetic and pharmacodynamic properties of oligonucleotide-based therapeutic agents.35 Oligonucleotide conjugates with lipophilic molecules have been shown to exhibit improved protein binding, nuclease resistance, and broader biodistribution and uptake in liver and jejunum in vivo relative to unconjugated controls.29,30,35-38 Cholesterol-conjugated siRNAs,29,30 antisense microRNAs (antagomirs),39 and antisense oligonucleotides40 enter cells in the absence of cationic lipids in vivo. Recently, a report demonstrated that siRNAs conjugated to polyspermine entered cells in the absence of transfection reagents in vitro.31 Conjugation of receptor-specific N-acetylgalactosamine (GalNAc),41-43 folic acid,44 or R-tocopherol45 to (27) Semple, S. C.; Akinc, A.; Chen, J.; Sandhu, A. P.; Mui, B. L.; Cho, C. K.; Sah, D. W. Y.; Stebbing, D.; Crosley, E. J.; Yaworski, E.; Hafez, I. M.; Dorkin, J. R.; Qin, J.; Lam, K.; Rajeev, K. G.; Wong, K. F.; Jeffs, L. B.; Nechev, L.; Eisenhardt, M. L.; Jayaraman, M.; Kazem, M.; Maier, M. A.; Srinivasulu, M.; Weinstein, M. J.; Chen, Q.; Alvarez, R.; Barros, S. A.; De, S.; Klimuk, S. K.; Borland, T.; Kosovrasti, V.; Cantley, W. L.; Tam, Y. K.; Manoharan, M.; Ciufolini, M. A.; Tracy, M. A.; de Fougerolles, A.; MacLachlan, I.; Cullis, P. R.; Madden, T. D.; Hope, M. J. Nat. Biotechnol. 2010, 28, 172–176. (28) Huh, M. S.; Lee, S.-Y.; Park, S.; Lee, S.; Chung, H.; Lee, S.; Choi, Y.; Oh, Y.-K.; Park, J. H.; Jeong, S. Y.; Choi, K.; Kim, K.; Kwon, I. C. J. Controlled Release 2010, 144, 134–143. (29) Soutschek, J.; Akinc, A.; Bramlage, B.; Charisse, K.; Constien, R.; Donoghue, M.; Elbashir, S.; Geick, A.; Hadwiger, P.; Harborth, J.; John, M.; Kesavan, V.; Lavine, G.; Pandey, R. K.; Racie, T.; Rajeev, K. G.; Roehl, I.; Toudjarska, I.; Wang, G.; Wuschko, S.; Bumcrot, D.; Koteliansky, V.; Limmer, S.; Manoharan, M.; Vornlocher, H.-P. Nature 2004, 432, 173–178. (30) Wolfrum, C.; Shi, S.; Jayaprakash, K. N.; Jayaraman, M.; Wang, G.; Pandey, R. K.; Rajeev, K. G.; Nakayama, T.; Charrise, K.; Ndungo, E. M.; Zimmermann, T.; Koteliansky, V.; Manoharan, M.; Stoffel, M. Nat. Biotechnol. 2007, 25, 1149–1157. (31) Nothisen, M.; Kotera, M.; Voirin, E.; Remy, J.-S.; Behr, J.-P. J. Am. Chem. Soc. 2009, 131, 17730–17731. (32) Minakuchi, Y.; Takeshita, F.; Kosaka, N.; Sasaki, H.; Yamamoto, Y.; Kouno, M.; Honma, K.; Nagahara, S.; Hanai, K.; Sano, A.; Kato, T.; Terada, M.; Ochiya, T. Nucleic Acids Res. 2004, 32, e109; DOI: 10.1093/nar/ gnh093. (33) Konate, K.; Crombez, L.; Deshayes, S.; Decaffmeyer, M.; Thomas, A.; Brasseur, R.; Aldrian, G.; Heitz, F.; Divita, G. Biochemistry 2010, 49, 3393–3402. (34) Rozema, D. B.; Lewis, D. L.; Wakefield, D. H.; Wong, S. C.; Klein, J. J.; Roesch, P. L.; Bertin, S. L.; Reppen, T. W.; Chu, Q.; Blokhin, A. V.; Hagstrom, J. E.; Wolff, J. A. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 12982– 12987. (35) Manoharan, M. Antisense Nucleic Acid Drug Dev. 2002, 12, 103– 128. (36) Jeong, J. H.; Mok, H.; Oh, Y.-K.; Park, T. G. Bioconjugate Chem. 2009, 20, 5–14. (37) Crooke, S. T.; Graham, M. J.; Zukerman, J. E.; Brooks, D.; Conklin, B. S.; Cummins, L. L.; Greig, M. J.; Guinosso, C. J.; Kornbrust, D.; Manoharan, M.; Sasmor, H. M.; Schleich, T.; Tivel, K. L.; Griffey, R. H. J. Pharmacol. Exp. Ther. 1996, 277, 923–237. (38) Rump, E. T.; de Vrueh, R. L.; Sliedregt, L. A.; Biessen, E. A.; van Berkel, T. J.; Bijsterbosch, M. K. Bioconjugate Chem. 1998, 9, 341–349. (39) Krutzfeldt, J.; Kuwajima, S.; Braich, R.; Rajeev, K. G.; Pena, J.; Tuschl, T.; Manoharan, M.; Stoffel, M. Nucleic Acids Res. 2007, 35, 2885– 2892. (40) Bijsterbosch, M. K.; Rump, E. T.; De Vrueh, R. L. A.; Dorland, R.; van Veghel, R.; Tivel, K. L.; Biessen, E. A. L.; van Berkel, T. J. C.; Manoharan, M. Nucleic Acids Res. 2000, 28, 2717–2725.

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FIGURE 1. Alkyne building blocks for postsynthetic CuAAC reaction on RNA.

antisense oligonucleotides, peptide nucleic acid, or siRNAs elicited receptor-mediated cellular uptake of the conjugated oligonucleotides in vitro or in vivo. However, synthesis of oligonucleotide conjugates is challenging due to lengthy synthetic procedures and frequent incompatibility with solid-phase oligonucleotide synthesis and deprotection conditions. Considering the enormous potential of oligonucleotideligand conjugates for delivering nucleic acid therapeutics to organs or tissues of interest, we evaluated click chemistry for synthesis of oligoribonucleotide/ligand conjugates. Three strategies were evaluated. In the first one, the oligonucleotide conjugate was obtained by solid-phase synthesis using modified building blocks which were obtained by click reaction at the monomer stage. In the second approach, solution-phase postsynthetic click conjugation of azido functionalized ligands to an alkyne-oligonucleotide scaffold bearing one or more alkyne moieties was evaluated. In the third strategy, azido-functionalized ligands were conjugated to a fully protected solid-support bound alkyne-oligonucleotide. We evaluated the effect of the physicochemical characteristics of the incoming azide on click conjugation with azidofunctionalized ligands derived from lipophilic long chain alkyls, cholesterol, oligoamine and carbohydrate. The siRNA conjugates synthesized using the CuAAC approach were evaluated for RNAi activity in vitro using a HeLa cell line stably transformed with the firefly and renilla luciferase gene. Results and Discussion Preparation of Nucleoside-Alkyne Building Blocks for Postsynthetic Oligonucleotide Conjugation. The nucleoside-alkyne building blocks shown in Figure 1 were selected for evaluating site-specific conjugation of small molecule ligands to siRNA under CuAAC reaction conditions. Alkynylation at the 20 -, 30 -, or 50 -hydroxyl of ribonucleosides (41) Hangeland, J. J.; Levis, J. T.; Lee, Y. C.; Ts’o, P. O. Bioconjugate Chem. 1995, 6, 695–701. (42) Biessen, E. A. L.; Sliedregt-Bol, K.; Hoen, P. A. C. T.; Prince, P.; Van der Bilt, E.; Valentijn, A. R. P. M.; Meeuwenoord, N. J.; Princen, H.; Bijsterbosch, M. K.; Van der Marel, G. A.; Van Boom, J. H.; Van Berkel, T. J. C. Bioconjugate Chem. 2002, 13, 295–302. (43) Hamzavi, R.; Dolle, F.; Tavitian, B.; Dahl, O.; Nielsen, P. E. Bioconjugate Chem. 2003, 14, 941–954. (44) Li, S.; Deshmukh, H. M.; Huang, L. Pharm. Res. 1998, 15, 1540– 1545. (45) Nishina, K.; Unno, T.; Uno, Y.; Kubodera, T.; Kanouchi, T.; Mizusawa, H.; Yokota, T. Mol. Ther. 2008, 16, 734–740.

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enables incorporation of one or more alkyne moiety to the desired position of an oligoribonucleotide for small molecule conjugation under CuAAC reaction conditions. The phosphoramidites 1-3 were designed to introduce alkyne functionality to the 50 -terminus of the oligonucleotide. The phosphoramidites 4 and 6 were chosen to incorporate the alkynes containing nucleosides at the 50 -terminal or internal positions of the oligonucleotide, whereas the solid supports 5 and 7 were selected for insertion of the alkyne containing nucleoside moiety at the 30 -terminus. The alkyne group was introduced via a carbamate linkage at the 50 position of uridine and 20 -O-methyluridine as shown in Scheme 1. Treatment of 20 ,30 -O-isopropylideneuridine (8) with phenyl chloroformate in anhydrous pyridine and subsequent treatment of the intermediate formed with propargylamine afforded compound 9 in >90% yield. The isopropylidene protection was removed by refluxing compound 9 in 80% acetic acid for 18 h to afford compound 10 in quantitative yield. Silylation of the hydroxyl group of compound 10 with tert-butyldimethylsilyl chloride (TBDMS-Cl) in anhydrous THF in the presence of pyridine and AgNO3 yielded a mixture of 20 - and 30 -O-TBDMS derivatives 11 and 11a (structure not shown in the Scheme), respectively. The isomeric ratio was approximately 7:2 (20 -O-TBDMS/30 -OTBDMS) as determined by 1H NMR analysis of the crude compounds. Chromatography on neutral alumina using 2% MeOH in CH2Cl2 as eluent produced good separation of the two isomers 11 and 11a. Phosphitylation of 20 -O-silylated nucleoside 11 afforded the phosphoramidite 1 in 71% isolated yield. The synthesis of phosphoramidite 2 was achieved with 45% overall yield from 30 -O-acetyl-20 -O-methyluridine (12)46 (Scheme 1). The precursor 14 for the synthesis of phosphoramidite 3 was obtained from compound 12 by Curtius rearrangement. Addition of diphenylphosphoryl azide to a mixture of compound 12 and 5-hexynoic acid in the presence of triethylamine in anhydrous DMF at ambient temperature and subsequent stirring at 100 °C for 18 h afforded compound 14 in 71% yield. Deacetylation of compound 14 using NaOMe in methanol at ambient temperature followed by phosphitylation yielded the desired phosphoramidite 3 in 31% overall yield. (46) Sekine, M.; Kurasawa, O.; Shohda, K.; Seio, K.; Wada, T. J. Org. Chem. 2000, 65, 3571–3578.

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Syntheses of 50 Alkyne-Functionalized Uridine Phosphoramiditesa

a Key: (i) (a) phenyl chloroformate, pyridine, rt, 3 h, (b) propargylamine, rt, 18 h, yield 9 (91%) and 13 (>95%); (ii) 80% AcOH in MeOH, reflux, 18 h, quant; (iii) AgNO3, pyridine, TBDMS-Cl, THF, rt, 18 h, 20%; (iv) 2-cyanoethyl N,N-diisopropylchlorophosphoramidite, N,N-diisopropylethylamine (DIEA), CH2Cl2, rt, 4 h, yields 1 (71%), 2 (70%) and 3 (70%); (v) diphenylphosphoryl azide, 5-hexynoic acid, Et3N, DMF, rt to 100 °C, 18 h, 71%; (vi) NaOMe, MeOH, rt, 2 h, yields 15 (67%) and 16 (65%).

The 20 - and 30 -alkyne-derived nucleosides were synthesized following modifications of the known procedures. 50 O-[Bis(4-methoxyphenyl)phenylmethyl] (50 -O-DMTr) derivatives 18 and 19 of 20 - and 30 -O-propargyl-5-methyluridine were directly synthesized from 50 -O-DMTr-5-methyluridine (17) by microwave-assisted alkylation (Scheme 2). The tinmediated alkylation of uridine with propargyl chloride in the presence of TBAI47 was optimized by replacing uridine with 50 -O-DMTr-uridine in a mixture of benzene and CH3CN. The microwave-assisted reaction was performed in a sealed bottle, and the reaction was complete within 4 h with a total isolated yield of about 70% (40% for 20 - and 32% for 30 -Opropargyl derivatives). The 50 -O-DMTr protection of the nucleoside remained intact and provided a good handle for purification after the tin-mediated alkylation. An attempt to alkylate 17 with propargyl chloride under non-microwave conditions resulted in partial deprotection of the 50 -O-DMTr group due to the extended alkylation time, and this made the isolation of the product difficult. The microwave-assisted propargylation of 50 -O-DMTr-uridine mediated by dibutyltin(IV) oxide was successful and is a promising approach for small-scale synthesis of 20 - and 30 -O-propargylated pyrimidines. The method was successfully extended to other ribonucleosides with protection of exocyclic amino groups (rABz, rCBz, and rGiBu) to give the corresponding 20 - and 30 -Opropargylated nucleosides in good yields (Table S1, Supporting Information). The phosphoramidite 6 was prepared as described above. Succinylation of 19 followed by coupling to (47) Srivastava, S. C.; Raza, S. K. PCT Int. Appl. 1995; p 46, WO 9518139.

long chain alkyl amine controlled pore glass support (lcaaCPG) under amide coupling conditions afforded the solid support 7 with 80 μM/g loading. Preparation of Small Molecules Carrying an Azido Group. The small molecules carrying azido groups shown in Figure 2 were chosen to evaluate the CuAAC reaction for highthroughput synthesis of oligonucleotide/ligand conjugates. The azides 20-23 are of fatty acid/alcohol origin to evaluate the CuAAC reaction between hydrophobic compounds and highly hydrophilic oligonucleotides in aqueous media. A more hydrophobic azide, 24, derived from cholest-5-en-3-ol(3β)-(6-hydroxyhexyl)carbamate48 was also evaluated. Amino azide 25 and its corresponding trifluoroacetamido-protected derivative 25a were synthesized from spermine (Scheme S2, Supporting Information) to evaluate the reaction of oligocationic small molecules with alkyne-functionalized oligonucleotides.49 Lastly, GalNAc derivatives 26 and 27 were chosen to evaluate whether hydrophilic small molecules would serve as effective reactants. GalNAc serves to deliver payloads to hepatocytes.42,50,51 (48) Medvedeva, D. A.; Maslov, M. A.; Serikov, R. N.; Morozova, N. G.; Serebrenikova, G. A.; Sheglov, D. V.; Latyshev, A. V.; Vlassov, V. V.; Zenkova, M. A. J. Med. Chem. 2009, 52, 6558–6568. (49) Manoharan, M.; Rajeev, K. G.; Butler, D.; Jayaraman, M.; Narayanannair, J. K.; Matsuda, S. PCT Int. Appl. 2009; p 243, WO 2009126933. (50) Rensen, P. C. N.; Sliedregt, L. A. J. M.; Ferns, M.; Kieviet, E.; van Rossenberg, S. M. W.; van Leeuwen, S. H.; van Berkel, T. J. C.; Biessen, E. A. L. J. Biol. Chem. 2001, 276, 37577–37584. (51) Varki, A., Cummings, R. D., Esko, J. D., Freeze, H. H., Stanley, P., Bertozzi, C. R., Hart, G. W., Etzler, M. E., Eds. Essentials of Glycobiology, 2nd ed.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 2009.

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JOC Featured Article SCHEME 2. Syntheses of 20 - and 30 -O-Propargyl-5-methyluridine Phosphoramidites and CPG Supportsa

a Key: (i) Bu2SnO (1.1 equiv), propargyl chloride (2.0 equiv), TBAI (0.5 equiv), benzene/CH3CN, microwave at 100 °C, yields 18 40% and 19 32%; (ii) 2-cyanoethyl N,N-diisopropylchlorophosphoramidite, DIEA, CH2Cl2, rt, 4-6 h, yield 6 51%; (iii) (a) succinic anhydride, DMAP, CH2Cl2, rt, 18 h, (b) HBTU, DIEA, lcaa-CPG (500 A˚, loading: 140 μM/g), DMF, 4 h.

Lipophilic azides52-54 20-23 (Figure 2) were synthesized from commercially available alcohols or bromides. Treatment of cholest-5-en-3-ol-(3β)-(6-hydroxyhexyl)carbamate48 (S17, Scheme S1, Supporting Information) with sodium azide in DMF afforded the azide 24 in 84% yield. The carbohydrate derivatives 26 and 27 were synthesized according to reported procedures.55 Synthesis of Click-Conjugated Monomers for Solid-Phase Oligonucleotide Synthesis. To demonstrate the click reaction at the monomer stage, we synthesized 50 -modified uridine 28 and 29 bearing linoleyl and GalNAc moieties, respectively. The conditions reported by Jatsch et al.56 were used (Scheme 3) for the synthesis. The reaction proceeded successfully with >85% isolated product yield. 20 to 30 TBDMS migration in MeOH/CH2Cl2 cosolvent was not observed during the reaction, presumably due to the slight acidity provided by [(CH3CN)4Cu]PF6 in CH2Cl2/MeOH. Phosphitylation of compounds 28 and 29 afforded the corresponding phosphoramidites 30 and 31 in 50 and 83% yields, respectively. The phosphoramidites 30 and 31 were subjected to standard oligonucleotide (52) Constantinou-Kokotou, V.; Kokotos, G.; Roussakis, C. Anticancer Res. 1998, 18, 3439–3442. (53) King, J. F.; Loosmore, S. M.; Aslam, M.; Lock, J. D.; McGarrity, M. J. J. Am. Chem. Soc. 1982, 104, 7108–7122. (54) Binder, W. H.; Kluger, C. Macromolecules 2004, 37, 9321–9330. (55) Gambert, U.; Lio, R. G.; Farkas, E.; Thiem, J.; Bencomo, V. V.; Lipt ak, A. Bioorg. Med. Chem. 1997, 5, 1285–1291. (56) Jatsch, A.; Kopyshev, A.; Mena-Osteritz, E.; Baeuerle, P. Org. Lett. 2008, 10, 961.

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synthesis and deprotection conditions to obtain modified oligonucleotides 46 and 56 with overall yield 60 and 64%, respectively, based on HPLC analysis. Conjugation of Ligands to Oligonucleotide in Solution Using the Click Reaction. To explore the validity of the CuAAC reaction for small-molecule conjugation to RNA we used alkyne-modified uridine derivatives. Since the sense strand of the model luciferase siRNA used lacked a uridine at the 50 -end, we decided to append the modified uridine monomers to the 50 - or 30 -end of the sense strand as an additional nucleotide. Solid-support bound alkyne-oligonucleotide scaffolds 32a-36a with a modified alkyne at the 50 -end, and the alkyne-oligonucleotide scaffolds 37a and 38a with alkynes at the 30 -end of the sense strand, shown in Table 1, were synthesized using the corresponding monomers according to the standard oligoribonucleotide synthesis conditions. Substitution of uridine at position 12 of the sense strand with 20 - and 30 -O-propargyluridine afforded the scaffolds 39a and 40a on the controlled pore glass support (CPG). Successive coupling of phosphoramidite 4 on CPG 5 followed by synthesis of the desired sequence on the support under standard oligoribonucleotide synthesis conditions afforded the solid-support bound oligonucleotide scaffold 41a with three 20 -O-propargyl-5-methyluridine moieties at the 30 -end of the sense strand (Table 1). An aliquot of each support-bound alkyne-oligonucleotide scaffold was deprotected under standard RNA deprotection conditions and analyzed by analytical HPLC and LC-MS to establish the integrity of each modified oligonucleotide synthesized (Table S2, Supporting Information). We used, in this study, the alkyne-oligonucleotides 32a-41a shown in Table 1 for evaluating the feasibility and usefulness of CuAAC reaction for small-molecule conjugation to siRNA. We first evaluated the CuAAC reaction for ligand conjugation to alkyne-oligonucleotide scaffolds in solution. In order to perform solution-phase CuAAC reactions on alkynefunctionalized oligonucleotides, the oligonucleotide scaffold 32 (Table S2, Supporting Information) was obtained from solid-support bound precursor oligonucleotide 32a (Table 1) by following standard oligonucleotide deprotection and purification protocols. The integrity of the propargylated oligonucleotide 32 was confirmed by LC-MS analysis. Reaction of purified alkyne-oligonucleotide 32 with excess unprotected azide 26a (GalNAcI-azide, Figure 2) in the presence of catalytic amounts of CuSO4 3 5H2O and 3 molar excess of sodium ascorbate (azide/CuSO4 3 5H2O/sodium ascorbate= 1:0.4:3 molar equiv) in MeOH/H2O (1:1, v/v) did not yield the desired conjugate 56 (data not shown). Significant degradation of the oligonucleotide 32 was observed both at room temperature overnight and at elevated temperature (60 °C) after 60 min. Presumably chelation of copper ions to the phosphate backbone and 20 -hydroxyl groups of unprotected oligonucleotide caused degradation. This was not unexpected as copper ion-mediated degradation of DNA oligonucleotides has been reported in the literature.57,58 At higher molar equivalents of CuSO4 (10 molar equiv) the reaction remained sluggish, and that made mass (57) Kanan, M. W.; Rozenman, M. M.; Sakurai, K.; Snyder, T. M.; Liu, D. R. Nature 2004, 431, 545–549. (58) Seela, F.; Pujari, S. S. Bioconjugate Chem. 2010, 21, 1629–1641.

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FIGURE 2. Azide monomers used for click reactions.

SCHEME 3.

Syntheses of 50 -Linoleyl and N-Acetylgalactosaminyl Uridine Click Monomersa

a Key: (i) 23 or 26, [(CH3CN)4Cu]PF6 (20 mol %), Cu powder (20 mol %), CH2Cl2/MeOH (4:1, v/v), rt, 4-18 h, yields 28 (98%) and 29 (88%); (ii) 2-cyanoethyl N,N-diisopropylchlorophosphoramidite, DIEA, CH2Cl2, rt, 4 h, yields 30 (50%) and 31 (83%).

spectral analysis of the product very difficult. After desalting, the chelated copper ion interfered with HPLC and LC-MS analysis. Treatment with EDTA prior to desalting did not remove copper ions completely from the product (data not shown). Use of powdered metallic copper25 under heating (60 °C, 30 min) resulted in quantitative reaction; however, the product was contaminated with copper ions. The observed m/z peak values corresponded to copper adducts (Figure S1, Supporting Information). Replacing hydrophilic azide 26a with hydrophobic 1-docosyl azide (20) in solution-phase conjugation to purified alkyne-oligonucleotide 32 in the presence of CuSO4 and sodium ascorbate was also not effective. Moreover, solvents compatible with the unpro-

tected highly hydrophilic oligonucleotide and hydrophobic alkyl azide that could facilitate an efficient azide-alkyne cycloaddition reaction were difficult to identify. Recently, copper wire has been successfully used as an efficient replacement for copper salt for the CuAAC reaction of 20 -alkyne/azido-modified adenosine with azides or alkynes respectively and also for the preparation of chimera of complementary oligonucleotides functionalized with an alkyne and an azide.12 The authors reported green coloration of the reaction mixture during prolonged stirring at 35 °C suggesting contamination/release of copper ion to the reaction mixture. Microwave-assisted CuAAC click conjugation has been successfully used for multiple labeling of solid-support J. Org. Chem. Vol. 76, No. 5, 2011

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Click Reaction of Alkyne-oligonucleotide Scaffolds with Small Molecule Azides 20-27

a The products 42-56 were obtained via CuAAC reaction of the corresponding azide and the alkyne-oligoribonucleotide precursor on solid support unless otherwise noted. All reactions were performed under microwave conditions except for compound 55. Typically, a heterogeneous mixture of the solid-support bound alkyne-oligoribonucleotide, the azide, CuSO4 3 5H2O, sodium ascorbate, and TBTA (1:3:0.4:3:3 molar equiv, respectively) in H2O/ MeOH/THF (1.2 mL, 2:2:1 v/v) was microwave irradiated at 60 °C in an Explorer-48 reactor for 45 min to obtain the corresponding conjugate. The 50 oligoamine-conjugated oligonucleotide 55 was prepared under thermal conditions (1.5 equiv of CuSO4 3 5H2O, 5 equiv of sodium ascorbate, 1:1 t-BuOH/ H2O, 50 °C, overnight). bThe oligonucleotides 46 and 56 were also directly synthesized from the phosphoramidite 30 and 31 with 60 and 64% overall yield, respectively, under solid-phase synthesis conditions. cReaction conversion was calculated from the ratio of product peak intensity to precursor peak intensity analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC). dReaction conversion was not determined due to difficulty in calculating the percentage conversion after solution-phase conjugation of the azide 26 with purified oligonucleotide 32 obtained after deprotection of 32a.

bound T12 DNA oligomer with carbohydrates.59 A variant of this approach in solution phase in the presence of tris(benzyltriazolylmethyl)amine (TBTA),21,60 a copper(I)-stabilizing agent, afforded the desired oligoribonucleotide conjugate 42. The microwave-assisted CuAAC reaction of the alkyl azide 20 and purified alkyne-oligonucleotide 32 in the presence of a large excess of CuSO4 3 5H2O (24 molar (59) Bouillon, C.; Meyer, A.; Vidal, S.; Jochum, A.; Chevolot, Y.; Cloarec, J.-P.; Praly, J.-P.; Vasseur, J.-J.; Morvan, F. J. Org. Chem. 2006, 71, 4700–4702. (60) Chan, T. R.; Hilgraf, R.; Sharpless, K. B.; Fokin, V. V. Org. Lett. 2004, 6, 2853–2855.

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equiv to alkyne), sodium ascorbate, and TBTA yielded the desired product 42. After irradiation for 45 min at 60 °C no starting material was detected by HPLC analysis (Figure S2, Supporting Information). The yields of the solution phase conjugations with and without microwave assistance were difficult to obtain reliably using HPLC methods due to incomplete reaction and degradation of the oligonucleotides. In addition, incompatible hydrophilic oligonucleotides and hydrophobic alkyl azide in a cosolvent in the presence of copper salt made the HPLC analysis more difficult. Furthermore, additional purification steps were required to remove TBTA and to minimize copper ion contamination from the product.

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Yamada et al. SCHEME 4.

CuAAC Reaction of Azides 20-26 with Alkyne-Oligoribonucleotide Scaffold 32a

(i) Alkyne/azide/CuSO4 3 5H2O/TBTA/sodium ascorbate (1:3:0.4:3:3 mol ratio), H2O/MeOH/THF (2:2:1, v/v), 1.2 mL total volume, microwave at 60 °C, 45 min, yields 42 (98%), 43 (95%), 44 (89%), 45 (96%), 46 (93%) and 55 (99%); (ii) standard oligonucleotide deprotection. Yield was calculated on the basis of RP-HPLC peak ratio of product to precursor.

TABLE 2. Optimization of CuAAC Reaction Conditions Using Azide 20 and Solid-Support Bound Fully Protected Alkyne-Oligonucleotide 32a solvent (2:2:1, v/v)b temp (°C) time (min) conversionc (%) reaction no. molar ratioa 1 2 3 4 5 6 7 8 9

1:16:0.4:3:0 1:16:0.4:3:3 1:3:0.4:3:3 1:3:0.1:3:3 1:3:0.4:3:3 1:3:0.4:3:3 1:3:0.4:3:3 1:3:0.4:3:3 1:3:0.4:3:3

H2O/MeOH/THF H2O/MeOH/THF H2O/MeOH/THF H2O/MeOH/THF H2O/MeOH/DMSO H2O/MeOH/DMF H2O/MeOH/THF H2O/MeOH/THF H2O/MeOH/THF

60 60 60 60 60 60 60 60 rt

45 45 45 45 45 45 45 45 48 h

no reaction 71 99 no reaction 88 79 57 60 quantitative

a Alkyne 32a/azide 20/CuSO4 3 5H2O/sodium ascorbate/TBTA. bThe total reaction volume is 1.2 mL except for reactions 7 and 8. For reaction 7 the volume was 0.24 mL and for 8 it was 3.6 mL. cReaction conversion was monitored by HPLC.

Conjugation of Ligands to Oligonucleotide on Solid Support Using Click Reaction. The conditions used by Morvan and co-workers for the synthesis of various carbohydrate-DNA conjugates by click reaction on solid-support 25,59,61 were optimized for fully protected support-bound alkyneoligoribonucleotide precursor 32a (Scheme 4, Table 1). The effects of molar equivalence of azide to alkyne, Cu catalyst, chelator (TBTA), temperature, and solvent for microwaveassisted CuAAC reaction were investigated; the findings are summarized in Table 2 and Figure S3, Supporting Information. The microwave-assisted CuAAC reaction of solid-support bound 32a with the azide 20 in the presence of TBTA afforded 71% yield of the desired conjugate 42 (Table 2, entry 2 and Figure S4, Supporting Information), whereas in the absence of TBTA no product was recovered (Table 2, entry 1). Addition of TBTA was required for the postsynthetic click reaction to occur on solid-support bound alkyneoligonucleotide. We found that 0.4 molar equiv of CuSO4 3 5H2O relative to the alkyne were required to achieve almost quantitative conversion to conjugate (Table 2, entry 3). Use of 3 molar equiv of the azide was sufficient for quantitative reaction. THF was the most effective cosolvent tested for dissolving lipophilic molecules in combination with water and methanol to produce higher yields rather than the use of DMF and/ or DMSO in place of THF (Table 2, entries 3, 5, and 6). Excessive amounts of lipophilic azides resulted in lower (61) Pourceau, G.; Meyer, A.; Vasseur, J.-J.; Morvan, F. J. Org. Chem. 2008, 73, 6014–6017.

product yields presumably due to aggregation of highly hydrophobic azide 20 (Table 2, entries 2 and 3). The reaction volume was also critical: a solvent volume of 1.2 mL was optimal for the Explorer-48 microwave reactor with lowest volume reaction vessel tested. Attempts to use less or more solvent (Table 2, reactions 7 and 8, total volume of solvent 0.24 and 3.6 mL, respectively) resulted in poor yield. The optimal reaction conditions (Table 2, entry 3) were microwave irradiation at 60 °C for 45 min with 1:3:0.4:3:3 molar ratios of alkyneoligonucleotide, azide, CuSO4 3 5H2O, TBTA, and sodium ascorbate in 1.2 mL of H2O, MeOH, and THF (2:2:1, v/v). The CuAAC reaction of 32a with the azide 20 also proceeded to completion in the absence of microwave irradiation in about 48 h at ambient temperature (Table 2, entry 9). HPLC and LC-MS analysis of the product obtained from microwave-assisted and unassisted reactions showed formation of the expected product. The observed rate enhancement of microwave-assisted conjugation reaction corroborated well with the cycloaddition reaction of small molecules.62,63 When the reaction was performed on a fully protected alkyneoligonucleotide on solid support, contamination of the product with copper ions was minimized to below the detection limit of our LC-MS analytical capability. Presumably poor availability of lone-pair electrons and lack of negative charges on the phosphate backbone for the fully protected oligonucleotide 32a minimized its coordination with the metal ion. (62) Appukkuttan, P.; Mehta, V. P.; Van der Eycken, E. V. Chem. Soc. Rev. 2010, 39, 1467–1477. (63) Kappe, C. O.; Dallinger, D. Mol. Diversity 2009, 13, 71–193.

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Conjugation of GalNAc to the Fully Protected Alkyne-Oligonucleotide 41aa

a (i) Alkyne/azide/CuSO4 3 5H2O/TBTA/sodium ascorbate 1:9:1.2:9:9 (molar ratio), H2O/MeOH/THF (2:2:1, v/v, vol. 1.2 mL), microwave at 60 °C, 45 min; (ii) standard oligonucleotide deprotection

TABLE 3. Gene Silencing Activity of siRNAs in Dual Luciferase Assay IC50b (nM) Tmc (°C) siRNA (S/ASa) 59/58 42/58 43/58 44/58 45/58 46/58 47/58 48/58 49/58 50/58 51/58 52/58 53/58 54/58

0.32 0.35 0.2 0.074 0.77 0.062 0.35 0.32 0.26 0.38 1.12 1.41 0.22 0.23

73.4 74.4 72.6 nd nd 74.4 nd nd nd nd 73.4 nd 72.5 69.6

a S and AS indicate sense and antisense strands, respectively; the unmodified duplex control was 59/58. See Table 1 for details of modified sense strands 42-54. bIC50 values were calculated from the doseresponse curves shown in Figure 3; all IC50 were determined in the presence of Lipofectamine 2000. c2 μM duplex concentration in 0.9% physiological saline solution; nd: not determined.

Our evaluation indicated that the microwave-assisted click reaction of solid-support bound alkyne-oligonucleotide scaffold with azide bearing ligands is more efficient and costeffective for small-scale oligonucleotide-conjugate synthesis than other strategies tested. The CuAAC reaction of solid-support bound alkyne-oligonucleotide also proceeded at ambient temperature in the absence of microwave irradiation; this procedure may prove useful with more reactive azide-bearing ligands than the ones used here. To further evaluate the scope of the click reaction on immobilized and protected alkyne-oligonucleotide scaffolds, different alkyl azides (21-24) were reacted with 32a on 1206

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solid-support under the optimized conditions. All expected products were obtained in excellent yields (Table 1, 43-46, Figure S5, Supporting Information). A typical HPLC profile of the deprotected precursor alkyne-oligonucleotide 32 and the product 46 after treatment with the linoleyl azide (23) indicated quantitative CuAAC reaction on the solid-support under the optimized conditions. The same 50 -conjugated oligonucleotide 46 was also synthesized from the phosphoramidite 30 using standard solid-phase oligonucleotide synthesis and deprotection conditions (Table 1). LC-MS data and HPLC profiles of the products obtained by both routes were identical. To evaluate the effect of the position of conjugation within the oligonucleotide, the linoleyl azide (23) was reacted with alkyne-oligonucleotide scaffolds carrying the alkyne moiety at 30 or 50 terminal positions or an internal position at either through the 20 or 30 position of the ribose moiety (Table 1, 32a-40a). The CuAAC reaction of oligonucleotide scaffolds modified with 20 -O-propargy-5-methylluridine (Table 1, 33a, 37a, and 39a) with azide 23 gave quantitative conversion to the expected products irrespective of the location of the modification on the oligonucleotide. Reaction with the 30 O-propargyluridine scaffolds 34a, 38a, and 40a gave slightly lower yields (∼90%) of the product (Table 1, 48, 52, and 54). Reaction with the oligonucleotide scaffold 36a modified with an alkyne at the 50 -terminus gave only 84% yield of the desired conjugate 50. The HPLC profiles of the purified products 47-54 are shown in Figure 5S, Supporting Information. The impact of physical properties of small molecule azides on CuAAC reaction on an immobilized oligonucleotidealkyne scaffold was evaluated. Hydrophilic and cationic small molecule azides were reacted with solid-support bound

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FIGURE 3. Plot of RNAi activity of various lipophile-conjugated luciferase siRNA via click chemistry at different positions on the sense strand in the Renilla luciferase mRNA expressed HeLa cell lines. The parent duplex is unmodified control siRNA duplex 59/58, where 58 (UCGAAGUACUCAGCGUAAGdTdT) and 59 (CUUACGCUGAGUACUUCGAdTdT) represent antisense and sense strands, respectively (IC50 data in Table 3). The sense strand of each siRNA conjugate was annealed with the unmodified antisense strand 58 to obtain the corresponding siRNA.

oligonucleotide-alkyne 32a under optimized CuAAC reaction conditions. Reaction with amino azide 25 did not yield the expected product, presumably due to chelation of copper ions to the unprotected amines. The click reaction of baselabile TFA-protected amino azide 25a with 32a yielded the desired product 55. The solid-support bound precursor 32a was reacted with compound 25a in the presence of CuSO4 3 5H2O (1.5 equiv) and sodium ascorbate (5 equiv) at 50 °C overnight to afford oligonucleotide conjugate 55 quantitatively. The efficiency of the reaction and the identity of the product formed were confirmed by HPLC and LC-MS analysis, respectively (Figure S6, Supporting Information). Previous studies have demonstrated that conjugation of multivalent GalNAc to drug results in binding of the conjugate with nM affinity to asialoglycoprotein receptor (ASGPR) and internalization into liver hepatocytes.64 We, therefore, explored the click reaction between GalNAc derivatives 27 carrying an azido group and an RNA oligonucleotide 41a bearing three alkyl functionalities at the 30 -end as shown in Scheme 5. Analysis of the crude product after deprotection showed that multiple incorporation of GalNAc onto solidsupport bound oligonucleotide was efficient. HPLC analysis of the deprotected crude product confirmed formation of 86% of the desired tri-GalNAc product 57 with insignificant amounts of mono- and di-GalNAc conjugate byproduct (Figure S7, Supporting Information). Dual-Luciferase Assay for RNA Interference Evaluation. A dual-luciferase assay was used to evaluate gene silencing by siRNAs formed from the modified oligonucleotides synthesized. A pGL4 vector containing the Renilla and firefly luciferase genes was used for the assay. The siRNA sequences were designed to target the firefly luciferase gene. siRNAs were transfected into cells using Lipofectamine2000. The gene silencing abilities of unmodified siRNA (59/58) and (64) Stockert, R. J. Physiol. Rev. 1995, 75, 591–609.

siRNA conjugates are summarized in Table 3; IC50 values were calculated from dose-response curves (Figure 3). The siRNAs 44/58 and 46/58 shown in Table 3 with the antisense strand 58 and complementary sense strands comprising of an added extra uridine derivative at the 50 -terminus conjugated to unsaturated lipid such as oleyl (C18, ω=1) or linoleyl (C18, ω = 2) (44, 46, Table 1) showed 4- to 5-fold improvement in IC50 over the control siRNA 59/58. The most active siRNA 46/58 had a Tm 1 °C higher than that of the control siRNA 59/58. In contrast, the siRNA 43/58 containing saturated stearyl conjugate 43 had an IC50 comparable to the unconjugated control. The siRNA formed from the 50 -cholesteryl conjugate (45) had a 2-fold lower IC50 than the control siRNA 59/58. Interestingly, replacement of the “5pU” sugar linker with ‘5pU2m’ reduced activity of the linoleyl conjugate (compare activities of 49/58 and 46/58, Table 3). The IC50’s calculated for “Lin-5pU” 46/58 and “Lin5pU2m” 49/58 were 0.062 and 0.26 nM, respectively. siRNAs constituted from strands with the linoleyl moiety conjugated through the 20 - (47) or 30 - (48) position of the ribosugar (“T2p” and “T3p” linkers, Tables 1 and 3) at the 50 -end of the sense strand showed comparable IC50s to the control siRNA 59/58. The 30 (“T3p”) and 20 (“T2p”) modified siRNAs had comparable activity in all cases: 47/58 vs 48/58, 51/58 vs 52/58, and 53/58 vs 54/58). Introduction of linoleyl to the 30 -end of the sense strand through either the “T2p” (51) or “T3p” (52) linker considerably reduced siRNA activity. Placement of the bulky residues “Lin-T2p” or “Lin-T3p” in the central region of the sense strand (53, 54) led to a small decrease in thermal stability (Tm) of the corresponding duplexes 53/58 and 54/58 compared to the control 59/58 (Table 3). In a recent study, we showed that local destabilization of the central region of the sense strand can enhance the potency of corresponding siRNA.65 Although in the current J. Org. Chem. Vol. 76, No. 5, 2011

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JOC Featured Article example, the linoleyl conjugates were also placed in the central region (position 12) of the sense strand, we only observed a minor improvement in potency. Presumably, the decrease in thermal stability was too small to have a significant effect on the potency of modified siRNAs. It also is interesting to note that insertion of an extra modified nucleotide to the 50 -end of the sense strand did not hamper the activity of the siRNA, whereas addition of a bulky nucleotide to the 30 -end of the sense strand was not well tolerated. Conclusions In this work, we developed and compared various approaches to conjugate small molecules with a range of physicochemical properties to oligoribonucleotides using click chemistry. We have demonstrated that the microwave-assisted CuAAC conjugation of azido-functionalized smallmolecule ligands to solid-support bound oligoribonucleotide allowed for efficient synthesis of oligoribonucleotideheteromolecule conjugates with the potential for high-throughput applications. The procedure described here allows rapid and economical synthesis of a wide variety of conjugates. Using the alkyne-oligonucleotide scaffolds described here, any azido-functionalized ligand can be introduced to a desired site within the oligonucleotide sequence and at selected positions on the sugar moiety (20 , 30 , or 50 position) or in the selected sites of nucleobase. Although we demonstrated the conjugation using uridine derivatives, the approach will be applicable to other nucleosides, employing the reported derivatization protocols.66-69 We have optimized the reaction conditions for conjugation of azides derived from hydrophobic, hydrophilic, and cationic compounds to alkyne-oligonucleotide scaffolds. Further optimization of this conjugation strategy for siRNA therapeutic screening with several ligands is in progress. The very recent demonstration of strain-promoted copper free click conjugation of ligands to RNA that is completely devoid of heavy metal ions is another promising approach for RNA oligonucleotide conjugation chemistry.70,71 However, because of simplicity of the chemistry involved and the versatility of the CuAAC reaction on solid-support bound alkyne-oligonucleotide, it is a promising high-throughput strategy for oligonucleotide conjugate synthesis for biological screening. Our laboratory is presently engaged in optimizing both copper-assisted72 (65) Addepalli, H.; Meena; Peng, C. G.; Wang, G.; Fan, Y.; Charisse, K.; Jayaprakash, K. N.; Rajeev, K. G.; Pandey, R. K.; Lavine, G.; Zhang, L.; Jahn-Hofmann, K.; Hadwiger, P.; Manoharan, M.; Maier, M. A. Nucleic Acids Res. 2010, 38, 7320–7331. (66) Egli, M.; Minasov, G.; Tereshko, V.; Pallan, P. S.; Teplova, M.; Inamati, G. B.; Lesnik, E. A.; Owens, S. R.; Ross, B. S.; Prakash, T. P.; Manoharan, M. Biochemistry 2005, 44, 9045–9057. (67) Manoharan, M.; Guinosso, C. J.; Cook, P. D. Tetrahedron Lett. 1991, 32, 7171–7174. (68) Manoharan, M.; Tivel, K. L.; Andrade, L. K.; Cook, P. D. Tetrahedron Lett. 1995, 36, 3647–3650. (69) Manoharan, M. Biochim. Biophys. Acta 1999, 1489, 117–130. (70) Jayaprakash, K. N.; Peng, C.-G.; Butler, D.; Varghese, J. P.; Maier, M. A.; Rajeev, K. G.; Manoharan, M. Org. Lett. 2010, 12, 5410–5413. (71) van Delft, P.; Meeuwenoord, N. J.; Hoogendoorn, S.; Dinkelaar, J.; Overkleeft, H. S.; van der Marel, G. A.; Filippov, D. V. Org. Lett. 2010, 12, 5486–5489. (72) Eltepu, L.; Peng, C. G.; Jayaprakash, K. N.; Yamada, T.; Jayaraman, M.; Kallanthottathil, G. R.; Manoharan, M. Abstracts of Papers. 240th ACS National Meeting; Aug 22-26, 2010, Boston, MA; American Chemical Society: Washington, DC, 2010; CARB-53.

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and copper-free70 azide-alkyne click reactions for synthesis of RNA conjugates. Experimental Section Materials. All reagents and solvents obtained from commercial suppliers were used without further purification. All reactions were carried out under argon in oven-dried glassware. Thinlayer chromatography was carried out on glass-backed silica-gel 60 F254 plates. Column chromatography was performed using silica gel (60 A˚, 230  400 mesh). Chemical shifts are given in parts per million (ppm); J values are given in hertz (Hz). All spectra were internally referenced to the appropriate residual undeuterated solvent. 20 ,30 -O-Isopropylidene-50 -O-propargylcarbamoyluridine (9). 20 ,30 -O-Isopropyrideneuridine (10.00 g, 35.18 mmol) in pyridine (350 mL) was treated with phenyl chloroformate (4.8 mL, 38.70 mmol) at room temperature for 2 h. Propargylamine (7.3 mL, 175.89 mmol) was added, and the reaction mixture was stirred at room temperature for 18 h. The reaction was monitored by TLC (Rf = 0.60; developing solvent CH2Cl2/MeOH, 12:1, v/v). Solvents and volatiles were removed in vacuo. The residue was suspended in CH2Cl2 (250 mL) and washed with satd aq NaHCO3 solution followed by standard workup. The residue was purified by flash chromatography on silica gel (98:2 CH2Cl2/MeOH, v/v) to obtain 9 (11.73 g, 91%) as a white foam: 1 H NMR (400 MHz, DMSO-d6) δ 11.41 (br, 1H), 7.73 (m, 1H), 7.65 (d, J = 8.0 Hz, 1H), 5.80 (d, J = 2.4 Hz, 1H), 5.62 (d, J = 8.0 Hz, 1H), 5.03 (dd, J = 6.4, 2.4 Hz, 1H), 4.74 (dd, J = 6.4, 3.1 Hz, 1H), 4.27-4.14 (m, 2H), 4.08 (m, 1H), 3.77 (m, 2H), 3.11 (m, 1H), 1.48 (s, 3H), 1.28 (s, 3H); 13C NMR (100 MHz, DMSOd6) δ 163.2, 155.5, 150.3, 142.5, 113.2, 101.8, 92.2, 84.1, 83.4, 81.2, 80.8, 73.1, 64.2, 29.8, 27.0, 25.1; MS calcd for C16H20N3O7 (MHþ) 366.13, found 366.12. 30 -O-Acetyl-20 -O-methyl-50 -O-propargylcarbamoyluridine (13). Compound 13 (5.03 g, 99%) was obtained as a white foam from compound 12 (4.00 g, 13.3 mmol), phenyl chloroformate (2.15 mL, 14.64 mmol), and propargylamine (3.21 mmol, 66.5 mmol) as described for the synthesis of 9. The residue after workup was purified by flash chromatography on silica gel (98:2 CH2Cl2/ MeOH, v/v) to obtain compound 13: 1H NMR (400 MHz, DMSO-d6) δ 11.49 (s, 1H), 7.91 (t, J = 5.7 Hz, 1H), 7.62 (d, J = 8.1 Hz, 1H), 5.87 (d, J = 6.6 Hz, 1H), 5.72 (d, J = 8.1 Hz, 1H), 5.22-5.16 (m, 1H), 4.26-4.12 (m, 4H), 3.85-3.79 (m, 2H), 3.28 (s, 3H), 3.18 (t, J = 2.3 Hz, 1H), 2.10 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 169.7, 162.9, 155.5, 150.6, 140.3, 102.7, 85.9, 81.2, 79.8, 79.4, 73.4, 70.5, 63.7, 58.2, 29.8, 20.6; MS calcd for C16H20N3O8 (MHþ) 382.12, found 382.00. 50 -O-Propargylcarbamoyluridine (10). Compound 9 (11.73 g, 32.10 mmol) in MeOH (20 mL) was refluxed with 80% AcOH (200 mL) for 18 h. The solution was concentrated in vacuo. The residue was coevaporated with methanol three times and then redissolved in 50 mL of anhydrous MeOH. The MeOH solution was sonicated for 30 min, and a white precipitate was obtained. The precipitate was filtered on a suction funnel and washed with CH2Cl2 (1 L). The white residue obtained was dried at 40 °C in a vacuum oven for 18 h to obtain compound 10 (10.34 g, 99%) 1 H NMR (400 MHz, DMSO-d6) δ 11.35 (br, 1H), 7.79 (m, 1H), 7.60 (d, J = 7.8 Hz, 1H), 5.78 (d, J = 5.5 Hz, 1H), 5.65 (d, J = 7.8 Hz, 1H), 5.42 (d, J = 5.5 Hz, 1H), 5.27 (d, J = 4.9 Hz, 1H), 4.33-3.72 (m, 7H), 3.13 (s, 1H); 13C NMR (100 MHz, DMSOd6) δ 172.1, 163.0, 155.7, 150.7, 140.7, 102.1, 87.8, 81.7, 81.2, 73.1, 72.5, 69.9, 64.0, 29.8; HRMS calcd for C13H16N3NaO7 (MNaþ) 348.0808, found 348.0812. 50 -O-Propargylcarbamoyl-20 -O-(tert-butyldimethylsilyl)uridine (11). Compound 10 (10.34 g, 28.30 mmol) in anhydrous THF (30 mL) was treated with silver nitrate (5.77 g, 34.00 mmol), pyridine (17.0 mL, 209.4 mmol), and TBDMS-Cl (4.27 g, 28.30 mmol)

Yamada et al. at room temperature for 18 h. The reaction mixture was diluted with CH2Cl2 (300 mL) and washed with water. The organic layer was dried over anhydrous Na2SO4 and then concentrated in vacuo. The residue was purified by chromatography on neutral alumina (98:2 CH2Cl2/MeOH, v/v) to obtain 11 (2.45 g, 20%, Rf = 0.15 on aluminum gel TLC) as a pure isomer: 1H NMR (400 MHz, DMSO-d6) δ 11.41 (br, 1H), 7.93-7.71 (m, 1H), 7.65 (d, J = 8.1 Hz, 1H), 5.83 (d, J = 5.1 Hz, 1H), 5.72 (dd, J = 8.1, 2.1 Hz, 1H), 5.23 (d, J = 6.0 Hz, 1H), 4.32-4.22 (m, 2H), 4.17 (m, 1H), 4.09-3.98 (m, 1H), 3.95-3.87 (m, 1H), 3.87-3.82 (m, 2H), 3.22-3.13 (m, 1H), 0.88 (d, J = 1.5 Hz, 12H), 0.09 (s, 3H), 0.07 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 163.4, 163.4, 156.1, 151.0, 140.7, 102.6, 88.4, 82.5, 81.7, 75.1, 73.7, 70.0, 64.2, 30.3, 26.2, 26.1, 18.5, 18.3, -4.1, -4.3, -4.6, -4.7; MS calcd for C19H29N3NaO7Si (MNaþ) 462.17, found 462.10. 50 -O-Propargylcarbamoyl-20 -O-(tert-butyldimethylsilyl)uridine30 -O-(N,N-diisopropyl)phosphoramidite (1). Compound 11a (452 mg, 1.02 mmol) in anhydrous CH2Cl2 (12 mL) was treated with DIEA (612 μL, 3.41 mmol) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (380 μL, 1.71 mmol) at room temperature for 6 h. The reaction mixture was diluted with CH2Cl2 (100 mL) and washed with brine (25 mL). The organic layer was dried over sodium sulfate and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (50:50:0.5 hexane/EtOAc/TEA, v/v) to obtain the phosphoramidite 1 (469 mg, 71%, Rf = 0.6) as a white foam: 1H NMR (400 MHz, DMSOd6) δ 11.51-11.33 (m, 1H), 7.96-7.71 (m, 1H), 7.71-7.53 (m, 1H), 5.91-5.79 (m, 1H), 5.76-5.62 (m, 1H), 4.39-4.08 (m, 5H), 3.873.53 (m, 6H), 3.20-3.05 (m, 1H), 2.85-2.69 (m, 2H), 1.28-1.04 (m, 12H), 0.92-0.72 (m, 9H), 0.16-0.10 (m, 6H); 31P NMR (162 MHz, DMSO-d6) δ 149.99, 148.44; HRMS calcd for C28H47N5O8PSi (MHþ) 640.2931, found 640.2927. 20 -O-Methyl-50 -O-propargylcarbamoyluridine-30 -O-(N,N-diisopropyl)phosphoramidite (2). The phosphoramidite 2 (1.18 g, 2.18 mmol, 87%, Rf = 0.1) was obtained as a white foam from compound 15 (1.00 g, 2.51 mmol) and 2-cyanoethyl N,Ndiisopropylchlorophosphoramidite (1.12 mL, 5.01 mmol) in the presence of DIEA (1.75 mL, 10.02 mmol) in anhydrous CH2Cl2 (25 mL) for 6 h as described above for synthesis of 1. The residue after workup was purified by flash chromatography on silica gel (3:7 hexane/EtOAc, v/v) to obtain the phosphoramidite 2: 1 H NMR (400 MHz, DMSO-d6) δ 11.43 (br, 1H), 7.95-7.74 (m, 1H), 7.72-7.52 (m, 1H), 5.98-5.76 (m, 1H), 5.76-5.58 (m, 1H), 4.40-3.90 (m, 5H), 3.86-3.47 (m, 6H), 3.33 (br, 3H), 3.18-2.84 (m, 1H), 2.83-2.70 (m, 2H), 1.28-1.00 (m, 12H); 31P NMR (162 MHz, DMSO-d6) δ 155.02, 154.38; HRMS calcd for C23H35N5O8P (MHþ) 540.2223, found 540.2220. 20 -O-Methyl-50 -O-(pentynyl)carbamoyluridine-30 -O-(N,N-diisopropyl)phosphoramidite (3). The phosphoramidite 3 (800 mg, 70%, Rf =0.2) was obtained as a white foam from compound 16 (750 mg, 2.03 mmol) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (878 μL, 2.76 mmol) in the presence of DIEA (1.38 mL, 7.52 mmol) in anhydrous CH2Cl2 (20 mL) for 18 h as described above for synthesis of 1. The residue after workup was purified by flash chromatography on silica gel (30:70:0.5 hexane/EtOAc/TEA, v/v) to obtain the phosphoramidite 3: 1H NMR (400 MHz, CD3CN) δ 9.04 (br, 1H), 7.58-7.45 (m, 1H), 5.97-5.85 (m, 1H), 5.85-5.73 (m, 1H), 5.73-5.57 (m, 1H), 4.46-4.12 (m, 4H), 3.95-3.58 (m, 5H), 3.57-3.36 (m, 3H), 3.32-3.09 (m, 2H), 2.82-2.65 (m, 2H), 2.26-2.17 (m, 2H), 2.16-2.13 (m, 1H), 1.83-1.61 (m, 2H), 1.31-1.11 (m, 12H); 31P NMR (162 MHz, CD3CN) δ 155.71, 155.47; HRMS calcd for C25H39N5O8P (MHþ) 568.2536, found 568.2550. 50 -O-(4,40 -Dimethoxytrityl)-30 -O-propargyl-5-methyluridine0 3 -O-(N,N-isopropyl)phosphoramidite (6). The phosphoramidite 6 (183 mg, 51%) was obtained as a white foam from compound

JOC Featured Article 19 (270 mg, 0.45 mmol) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (200 μL, 0.88 mmol) in the presence of DIEA (325 μL, 1.75 mmol) in anhydrous CH2Cl2 (3 mL) for 6 h as described above for the synthesis of 1. The residue after workup was purified by flash chromatography on silica gel (4:6 hexane/EtOAc, v/v) to obtain the phosphoramidite 6: 1H NMR (400 MHz, DMSO-d6) δ 11.42-11.41 (m, 1H), 7.60-7.17 (m, 10H), 6.97-6.79 (m, 4H), 5.92-5.88 (m, 1H), 4.67-4.58 (m, 1H), 4.46-4.24 (m, 3H), 4.26-3.94 (m, 2H), 3.76 (m, 7H), 3.56 (m, 3H), 3.40-3.14 (m, 2H) 2.77-2.69 (m, 2H), 1.50-0.84 (m, 15H); 31P NMR (162 MHz, DMSO-d6) δ 156.06, 154.99; MS calcd for C43H51N4NaO9P (MNaþ) 821.32, found 821.30. 2 0 -O-TBDMS-5 0 -O-[1-linoleyl-(1,2,3-triazo-3-yl)methyl]carbamoyluridine-30 -O-(N,N-diisopropyl)phosphoramidite (30). The phosphoramidite 30 (463 mg, 50%) was obtained as a white foam from compound 28 (730 mg, 1.00 mmol) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (335 μL, 1.50 mmol) in the presence of DIEA (525 μL, 3.00 mmol) in anhydrous CH2Cl2 (10 mL) for 4 h as described above for the synthesis of 1. The residue after workup was purified by flash chromatography on silica gel (60:30:0.5 hexane/EtOAc/TEA, v/v) to obtain the phosphoramidite 30: 1H NMR (400 MHz, DMSO-d6) δ 11.43-11.42 (1H, br), 7.91-7.89 (2H, m), 7.66-7.59 (1H, m), 5.86-5.84 (1H, m), 5.70-5.66 (1H, m), 5.38-5.27 (3H, m), 4.32-4.13 (8H, m), 3.83-3.56 (4H, m), 2.80-2.72 (4H, m), 2.04-1.99 (3H, m), 1.79-1.76 (2H, m), 1.33-1.11 (30H, m), 0.87-0.82 (12H, m), 0.05-0.02 (6H, m); 31P NMR (162 MHz, DMSO-d6) δ 149.98, 148.37; MS calcd for C46H79N8NaO8PSi (MNaþ) 953.54, found 953.40. Compound 31. The phosphoramidite 31 (403 mg, 83%) was obtained as a white foam from compound 29 (400 mg, 0.44 mmol) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (150 μL, 0.67 mmol) in the presence of DIEA (230 μL, 1.33 mmol) in anhydrous CH2Cl2 (4 mL) for 4 h as described above for the synthesis of 1. The residue after workup was purified by flash chromatography on silica gel (EtOAc then 2:98:0.5 MeOH/CH2Cl2/TEA, v/v) to obtain the phosphoramidite 31: 1 H NMR (400 MHz, DMSO-d6) δ 11.37 (br, 1H), 7.94-7.88 (m, 1H), 7.89-7.76 (m, 2H), 7.65-7.58 (m, 1H), 5.81-5.72 (m, 1H), 5.68-5.58 (m, 1H), 5.25-5.18 (m, 2H), 4.99-4.92 (m, 1H), 4.57-4.43 (m, 3H), 4.30-4.11 (m, 5H), 4.05-3.98 (m, 4H), 3.97-3.84 (m, 2H), 3.84-3.67 (m, 3H), 3.64-3.44 (m, 3H), 2.16-2.05 (m, 3H), 2.05-1.93 (m, 3H), 1.93-1.82 (m, 3H), 1.82-1.74 (m, 3H), 0.91-0.71 (m, 10H), 0.09-0.05 (m, 6H); 31P NMR (162 MHz, DMSO-d6) δ 148.98, 148.37; MS calcd for C46H74N9NaO18PSi (MNaþ) 1123.17, found 1123.30. 30 -O-Acetyl-20 -O-methyl-50 -O-(4-pentynyl)carbamoyluridine (14). 5-Hexynoic acid (3.11 mL, 28.1 mmol) in DMF (150 mL) was treated with diphenylphosphoryl azide (10.7 mL, 56.2 mmol) and triethylamine (8.12 mL, 56.2 mmol) at ambient temperature to 100 °C for 6 h. Then 30 -O-acetyl-20 -O-methyluridine (12, 2.0 g, 7.04 mmol) was added to the reaction mixture, and stirring was continued at 100 °C for 18 h. Progress of the reaction was monitored by TLC. The reaction mixture was cooled to ambient temperature, diluted with EtOAc, and washed with brine. The organic layer was dried over sodium sulfate and then concentrated in vacuo. The residue was purified by flash chromatography on silica gel (98.5:1.5 CH2Cl2/MeOH, v/v) to obtain 14 (2.07 g, 71%, Rf = 0.2) as a white foam: 1H NMR (400 MHz, DMSOd6) δ 11.49 (s, 1H), 7.64 (d, J = 8.1 Hz, 1H), 7.44 (t, J = 5.6 Hz, 1H), 5.87 (d, J = 6.4 Hz, 1H), 5.72 (d, J = 8.1 Hz, 1H), 5.26-5.09 (m, 1H), 4.28-4.04 (m, 4H), 3.28 (s, 3H), 3.11-2.96 (m, 2H), 2.79 (t, J = 2.5 Hz, 1H), 2.21-2.11 (m, 2H), 2.13 (s, 3H), 1.61-1.46 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ 169.6, 162.95, 155.7, 150.6, 140.3, 102.7, 86.15, 83.9, 79.8, 79.5, 71.5, 70.5, 63.3, 58.2, 28.3, 20.6, 15.2; MS calcd for C18H23N3O8 (MHþ) 410.15, found 410.00. J. Org. Chem. Vol. 76, No. 5, 2011

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JOC Featured Article 20 -O-Methyl-50 -O-propargylcarbamoyluridine (15). Compound 13 (4.35 g, 14.49 mmol) was treated with 0.5 M NaOMe in methanol (200 mL) for 4 h. The solution was concentrated in vacuo. The residue was purified by flash chromatography on silica gel (95:5 CH2Cl2/MeOH, v/v) to obtain 15 (2.75 g, 67%, Rf = 0.3) as a white foam: 1H NMR (400 MHz, DMSO-d6) δ 11.41 (br, 1H), 7.97 (dd, J = 5.7 Hz, 1H), 7.60 (d, J = 8.1 Hz, 1H), 5.87 (d, J = 5.5 Hz, 1H), 5.68 (d, J = 8.1 Hz, 1H), 5.37 (d, J = 5.8 Hz, 1H), 4.28-4.02 (m, 3H), 4.04-3.95 (m, 1H), 3.91-3.84 (m, 1H), 3.84-3.75 (m, 2H), 3.46 (s, 3H), 3.21 (dd, J = 2.2 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ 163.0, 155.6, 150.5, 140.4, 102.3, 86.0, 82.1, 81.4, 81.3, 73.3, 68.6, 63.9, 57.5, 29.8; MS calcd for C14H18N3O7 (MHþ) 340.11, found 340.10. 20 -O-Methyl-50 -O-(4-pentynyl)carbamoyluridine (16). Compound 14 (296 mg, 0.72 mmol) was treated with 7 N NH3 in methanol (5 mL) at ambient temperature for 4 h. The solution was concentrated in vacuo and then coevaporated with CH2Cl2 to remove traces of methanol. The residue was dissolved in a minimal amount of EtOAc/hexane/CH2Cl2 and sonicated for 5 min to give a white precipitate. The precipitate was filtered, washed with hexane, and dried over a suction funnel. Drying in a vacuum oven at 40 °C for 18 h gave compound 16 (173 mg, 65%): 1H NMR (400 MHz, DMSO-d6) δ 11.41 (s, 1H), 7.61 (d, J = 8.1 Hz, 1H), 7.36 (t, J = 5.5 Hz, 1H), 5.86 (d, J = 5.4 Hz, 1H), 5.67 (d, J = 8.1 Hz, 1H), 5.35 (d, J = 5.9 Hz, 1H), 4.26-3.81 (m, 5H), 3.35 (s, 3H), 3.13-2.98 (m, 2H), 2.78 (t, J = 2.4 Hz, 1H), 2.22-2.08 (m, 2H), 1.69-1.49 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ 162.9, 155.8, 150.5, 140.4, 102.2, 86.1, 83.9, 82.2, 81.5, 71.4, 68.6, 63.5, 57.5, 28.3, 15.2; MS calcd for C16H22N3O7 (MHþ) 368.14, found 368.3; HRMS calcd for C16H21N3NaO7 (MNaþ) 390.1277, found 390.1283. General Procedure for the Propargylation of the 20 /30 -Hydroxyl Group of Ribonucleoside Derivatives. 50 -O-DMTr-nucleoside (1.00 molar equiv), dibutyltin(II) oxide (1.10 molar equiv), tetrabutylammonium iodide (0.50-2.00 molar equiv), and propargyl chloride (2.00 molar equiv) were added to the solution of benzene/CH3CN (1:1, 0.2 M). The suspension was microwave irradiated at 100 °C for 1-6 h. Aqueous workup followed by flash silica gel chromatography gave the desired propargylated nucleosides. 50 -O-(4,40 -Dimethoxytrityl)-30 -O-propargyl-5-methyluridine (19): 1 H NMR (400 MHz, DMSO-d6) δ 11.37 (br, 1H), 7.59-7.18 (m, 10H), 6.91 (m, 4H), 5.80-5.69 (m, 1H), 5.63 (d, J = 5.99 Hz, 1H), 4.46-4.20 (m, 4H), 4.18-3.99 (m, 1H), 3.72 (s, 6H), 3.56-3.46 (m, 1H), 3.27-3.15 (m, 2H), 1.37 (d, J = 0.8 Hz, 3H); 13C NMR (100 MHz, DMSO-d6) δ 163.6, 158.2, 158.2, 150.6, 144.6, 135.5, 135.3, 135.0, 129.7, 128.0, 127.6, 126.8, 113.3, 109.5, 87.9, 86.0, 80.6, 80.1, 77.6, 76.0, 72.3, 62.9, 58.0, 55.1, 31.0, 22.1, 14.0, 11.6; HRMS calcd for C34H34N2NaO8 (MNaþ) 621.2213, found 621.2214. 20 -O-(tert-Butyldimethylsilyl)-50 -O-[1-linoleyl(1,2,3-triazo-3-yl)methyl]carbamoyluridine (28). Compound 11 (100 mg, 0.17 mmol) in CH2Cl2/MeOH (4:1, v/v) (2 mL) was treated with 23 (59 mg, 0.17 mmol), tetrakis(acetonitrile)copper(I) hexafluorophosphate (13 mg, 0.03 mmol), and copper (2 mg, 0.03 mmol) at ambient temperature for 3 h. The solution was concentrated in vacuo. The residue was purified by flash chromatography on silica gel (1:3 hexane/EtOAc, v/v) to obtain 28 (122 mg, 98%) as a white foam: 1H NMR (400 MHz, DMSO-d6) δ 11.35 (s, 1H), 7.91 (s, 1H), 7.82 (br, 1H), 7.60 (d, J = 7.8 Hz, 1H), 5.78 (d, J = 5.0 Hz, 1H), 5.63 (d, J = 7.8 Hz, 1H), 5.46-5.24 (m, 4H), 5.17 (br, 1H), 4.35-4.16 (m, 6H), 4.16-4.08 (m, 1H), 4.06-3.96 (m, 1H), 3.93-3.82 (m, 1H), 2.73 (dd, J = 6.2 Hz, 2H), 2.16-1.90 (m, 2H), 1.90-1.66 (m, 2H), 1.60-1.03 (m, 16H), 1.04-0.54 (m, 12H), 0.33-0.30 (m, 9H); 13C NMR (100 MHz, DMSO-d6) δ 161.7, 154.7, 139.1, 128.6, 128.5, 126.6, 101.0, 86.7, 80.9, 73.6, 68.4, 62.3, 48.1, 39.0, 38.8, 38.6, 38.0, 37.8, 34.8, 29.7, 28.6, 27.8, 27.5, 27.4, 27.2, 25.5, 25.4, 24.7, 24.4, 24.0, 1210

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Yamada et al. 20.8, 16.7, 12.7, -6.0, -6.4; MS calcd for C37H63N6O7Si (MHþ) 730.45, found 731.20. Compound 29. Compound 11 (500 mg, 1.14 mmol) in CH2Cl2/ MeOH (4:1, v/v) was treated with 26 (460 mg, 1.14 mmol), tetrakis(acetonitrile)copper(I) hexafluorophosphate (42 mg, 0.11 mmol), and copper (7 mg, 0.11 mmol) at room temperature for 18 h. The solution was concentrated in vacuo. The residue was directly loaded on flash silica gel column without aqueous workup and purified (eluent: 9:1 CH2Cl2/MeOH, v/v) to obtain 29 (800 mg, 88%) as a white foam: 1H NMR (400 MHz, DMSOd6) δ 11.37 (s, 1H), 7.90 (s, 1H), 7.84 (dd, J = 14.7, 7.6 Hz, 2H), 7.61 (d, J = 8.1 Hz, 1H), 5.78 (d, J = 5.0 Hz, 1H), 5.64 (d, J = 8.0 Hz, 1H), 5.25-5.18 (m, 2H), 4.96 (dd, J = 11.2, 3.3 Hz, 1H), 4.57-4.43 (m, 3H), 4.30-4.11 (m, 5H), 4.05-3.98 (m, 4H), 3.97-3.84 (m, 2H), 3.84-3.67 (m, 3H), 3.64-3.44 (m, 2H), 2.09 (s, 3H), 1.99 (s, 3H), 1.89 (s, 3H), 1.76 (s, 3H), 0.83 (s, 9H), 0.03 (d, J = 11.8 Hz, 6H); 13C NMR (100 MHz, DMSO-d6) δ 170.0, 169.9, 169.6, 169.3, 162.9, 155.9, 150.5, 144.8, 140.2, 123.1, 102.1, 100.9, 87.9, 82.0, 74.7, 70.4, 69.9, 69.5, 69.2, 68.8, 68.2, 66.7, 63.5, 61.4, 49.3, 25.6, 22.8, 20.49, 20.42, 20.41, 17.8, -4.8, -5.2; HRMS calcd for C37H58N7O17Si (MHþ) 900.3658, found 900.3660. Loading of 50 -O-(4,40 -Dimethoxytrityl)-30 -O-propargyl-5methyluridine on CPG Support (7). Compound 19 (286 mg, 0.48 mmol) in anhydrous CH2Cl2 (50 mL) was treated with succinic anhydride (231 mg, 0.68 mmol) and DMAP (111 mg, 0.91 mmol) at ambient temperature for 18 h. The solution was concentrated under reduced pressure. The residue was chromatographed on silica (eluent: TEA/MeOH/CH2Cl2, 5:5:90) to obtain the pure succinate (383 mg, 0.49 mmol, 99%, Rf = 0.6). The succinate was dissolved in anhydrous DMF (50 mL), and DIEA (320 μL, 1.84 mmol) and HBTU (191 mg, 0.50 mmol) were added. The solution was agitated briefly for 2 min, lcaa-CPG (pore size 500 A˚ NH2, loading of 140 μmol/g, 3.57 g) was added to the reaction mixture, and the slurry was agitated on a wristaction shaker for 4 h at ambient temperature. The CPG was filtered, washed with CH2Cl2/MeOH (9:1, v/v, 400 mL), and dried over suction funnel for 15 min. The remaining amino residues on the CPG were capped by pyridine/Ac2O/TEA (75:25:5, v/v, 100 mL) treatment for 15 min. The CPG was filtered, washed with CH2Cl2/MeOH (9:1, v/v, 600 mL), and then dried over a suction funnel. Finally, the CPG was dried under vacuum at ambient temperature for 18 h to give 7 (4.08 g CPG, loading: 80 μmol/g). General Procedure for Solid-Phase Synthesis of Modified siRNA Targeting the Firefly Luciferase Gene. Oligonucleotide sequences bearing alkyne moieties at desired sites (30 or 50 termini or internal position, Table 1) and other functional groups on the 50 -terminal position (entry 46 and 56 in Table 1) were synthesized on an ABI 394 DNA synthesizer using standard phosphoramidite chemistry with commercially available 50 -O-(4,40 -dimethoxytrityl)-30 -O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite monomers of uridine (U), 4-N-acetylcytidine (CAc), 6-N-benzoyladenosine (ABz), and 2-N-isobutyrylguanosine (GiBu), 20 -O-tert-butyldimethylsilyl-protected phosphoramidites, and 50 -O-(4,40 -dimethoxytrityl)-20 -deoxythymidine30 -O-(2-cyanoethyl-N,N-diisopropyl) phosphoramidite (dT). Extended nucleotide coupling times (30 min) were applied at steps incorporating modified phosphoramidites (alkyne monomers 1, 2, 3, 4, 6, and triazoyl monomers 30 and 31). After synthesis, a small portion of the oligonucleotide bound to CPG was treated with 100 μL of methylamine solution (40 wt % in water) in a 1 mL microtube at 65 °C for 10 min. The mixture was cooled on dry ice for 5 min and the solid suspension was spun down. Of the supernatant, 80 μL was decanted into another microtube and heated with 120 μL of TEA 3 3HF at 65 °C for 12 min. The purity of crude oligonucleotides was analyzed by RP-HPLC or anion-exchange high-performance

Yamada et al. liquid chromatography (IEX-HPLC) and the mass was confirmed by LC-MS analysis prior to postsynthetic CuAAC reaction. General Procedure for Click Reaction on Solid Support. To a 0.6 μmol solid-supported alkyne-RNA phosphotriester was added a mixture of an azide (3 equiv by alkyne, 1.8 μmol, 36 μL of a 50 mM solution in THF), freshly prepared CuSO4 3 5H2O (0.4 equiv, 0.24 μmol, 5 μL of a 50 mM solution in H2O), freshly prepared sodium ascorbate (3 equiv, 1.8 μmol, 37 μL of a 50 mM solution in H2O), and TBTA (3 equiv, 1.8 μmol, 37 μL of a 50 mM solution in THF). Water/MeOH/THF (2:2:1 v/v) was added to obtain a total volume of 1200 μL. The resulting preparation was heated in a sealed glass tube in an Explorer48 microwave synthesizer at a 100 W and a 30 s premixing time. The temperature was monitored with an internal infrared probe and held at 60 °C over 45 min. The solution was removed, and the CPG supports were washed with THF and MeOH then dried. The product on the CPG supports were cleaved and deprotected by treating the solid-support with 100 μL of methylamine solution (40 wt % in water) at 65 °C for 10 min. The mixture was cooled on dry ice for 5 min and the solid suspension was spun down. Of the supernatant, 80 μL was decanted into another microtube and heated with 120 μL of TEA 3 3HF at 65 °C for 12 min. The product was analyzed by LC-MS and by RP-HPLC (C4 column, 150  3.9 mm i.d., 5 μm, 300 A˚) using a linear gradient of 0 to 70% B over 24 min at 30 °C at a flow rate of 1 mL/min (buffer A: 50 mM TEAA, pH 7.0; buffer B: CH3CN). Completion of the CuAAC reaction was estimated by comparing the purity of starting material alkyne-RNAs and their corresponding triazole products by RP-HPLC. After base and sugar deprotection, oligonucleotides were purified by RPHPLC with C4 column (300  7.8 mm i.d., 15 μm, 300 A˚) using a linear gradient of 0 to 90% B in 40 min at room temperature at a flow rate of 3 mL/min (buffer A: 50 mM TEAA, pH 7.0; buffer B: CH3CN). Duplex Annealing. For duplex annealing, 1 mM stock solutions of oligonucleotides were prepared in deionized water. For the cell culture experiments, 0.05 mM stock solutions of siRNA duplexes were prepared by mixing equimolar amounts of complementary sense and antisense strands in PBS buffer. The solutions were heated at 95 °C for 5 min and then allowed to slowly cool to room temperature. To ensure low variability in duplex concentration, the optical densities of the final duplex

JOC Featured Article solutions in PBS buffer were measured and the duplex preparation was repeated if the optical density was not within a 10% range of a reference sample (parent duplex). Cell Culture. HeLa SS6 cells, stably transfected with the luciferase plasmids, were grown at 37 °C in 5% CO2 in Dulbecco0 s modified Eagle0 s medium supplemented with 10% fetal bovine serum (FBS) and 0.5 mg/mL Zeocin and 0.5 μg/mL puromycin (selective for cells transfected with plasmids). The cells were maintained in exponential growth phase. Luciferase Assay. The cells were plated in 96-well plates (0.1 mL medium per well) and were at about 90% confluence at transfection. The cells were grown for 24 h, and the culture medium was changed to Opti-MEM, 0.5 mL per well. Transfection of siRNAs was carried out with Lipofectamine 2000 as described by the manufacturer for adherent cell lines at siRNA concentrations ranging from 0.002 to 8.0 nM. The final volume was 150 μL per well. The cells were harvested 24 h post transfection and lysed using passive lysis buffer (PLB), 100 μL per well. The luciferase activities of the samples were measured using a Victor FL Luminometer with 20 μL of sample and 75 μL each of Luciferase assay reagent II and Stop & Glo reagent. The inhibitory effects of the siRNAs were expressed as normalized ratios between the activities of the reporter (firefly) luciferase gene and the control (Renilla) luciferase gene relative to untreated controls (no siRNA, but with Lipofectamine 2000). Values represent the mean of triplicates. The potency of the siRNAs was determined by calculating the IC50 values from dose-response curves using XL-Fit software.

Acknowledgment. T.Y. acknowledges the Genome Network Project from the Ministry of Education, Culture, Sports, Science and Technology, Japan, for a fellowship. Supporting Information Available: Yields of 20 /30 -O-pro-

pargylated 50 -O-DMTr nucleosides under optimized microwave-assisted reaction conditions; synthetic scheme of alkyl azides 20-24, scheme and synthetic procedures for azides 25 and 25a; NMR spectra of nucleoside derivatives and azides 24, 25, and 25a; LC-MS characterization of alkyne-oligonucleotide scaffolds 32a-41a, and HPLC profiles of click reaction of alkyne-oligonucleotides with azides and purified oligonucleotide conjugates. This material is available free of charge via the Internet at http://pubs.acs.org.

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